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1. PBZ SA A F A 01 3 5 7 0 1 2 3 B OsAP77 OsAP77 Actin Actin B A G M. oryzae (vir) B INA +/- -/- +/- +/+ +/+ +/+ 0 1 2 3 01 3 5 7 W1 W2 1 2 3 4 5 6 7 8 9 H2O OsAP77 OsAP77 A OsAP77 Actin Actin H2O2 Tos17 M. oryzae (avr) B C H D 012 24 48 72 0 1 2 3 C C OsAP77 C C OsAP77 D OsAP77 Actin Actin Actin D D X. oryzae pv. oryzae I ABA 01 3 5 7 0 1 2 3 OsAP77 OsAP77 Actin Actin MeJA Wound J E 0 1 2 3 06 12 36 48 OsAP77 OsAP77 Actin Actin 3. Effects of signaling molecules 3. RT-PCR analysis of OsAP77 Ｉｎｔｒｏｄｕｃｔｉｏｎ A rice aspartic protease 77 gene (OsAP77, Os10g0537800, accession number AK061277) is one of probenazole (PBZ) inducible genes (Shimono et al, 2003).Since PBZ is a disease resistance inducer, it is expected thatＯｓＡＰ７７may be involved in resistance against pathogen infection.Therefore, we investigated the role ofＯｓＡＰ７７ in disease resistance. Ｍａｔｅｒｉａｌsand　Ｍｅｈｔｏｄｓ Transgenic plants: GUS gene was located downstream of the promoter region of OsAP77. Transgenic rice was produced to express GUS. Inoculation: Strains of blast fungus Magnaporthe oryzae, virulent race 001, virMo (MAFF #238988) and avirulent race 102.0, avrMo (MAFF #238991), were obtained from NIAS Genebank, (http://www.gene.affrc.go.jp/index_en.php). Xanthomonas oryzae pv. oryzae strain 001 (MAFF #311018, NIAS Gene Bank) was used. Chemical treatments: Rice seedlings at 4-leaf stage were dipped in 3 ml each of solutions containing 10 mM SA, 5 mM INA, 20 mM H2O2, 10 mM ABA 10 mM methyl jasmonate (MeJA) and incubated for 72 h in the growth. OsAP77 mutant rice: OsAP77 mutant line (NC2562) was obtained from National Institute of Agrobiological Sciences (NIAS, https://tos.nias.affrc.go.jp/~miyao/pub/tos17/). Through the microarray analysis in rice treated by probenazole (PBZ), a chemical inducer of disease resistance, we previously reported that an aspartic protease (OsAP77) gene was PBZ-inducible. To elucidate the role(s) of this gene in defense response against pathogens, we took two approaches; one is to use transgenic rice plants harboring the chimeric gene consisting of the promoter of OsAP77 and the structural gene encoding β-glucuronidase (GUS) reporter and the other is to use the gene-knockout (KO) mutant line by the insertion of a rice retrotransposon, Tos17. Histochemical analysis showed that GUS activity was specifically detected in the vascular tissues of the transgenic leaves. The GUS activity in the vascular tissues was remarkably induced not only by the infection with Magneporthe oryzae or Xanthomonas oryzae pv. oryzae, but also by signaling molecules that have roles in defense and stress response, such as salicylic acid, isonicotinic acid, hydrogen peroxide or abscisic acid. The KO mutant line of OsAP77 was tested for infection by M. oryzae or X. oryzae pv. oryzae. The symptom severity was compared among three groups of plants; homozygous, heterozygous and wild type plants for OsAP77 mutation. As a result, homozygous plants showed the most severe symptoms. These results suggest that OsAP77 has a positive role in defense response against pathogens. This work was supported by the Program for Promotion of Basic and Applied Researches in Bio-oriented Industry (BRAIN). ＡＢＳＴＲＡＣＴ ACKNOWLEDGEMENTS This work was supported by the Program for Promotion of Basic and Applied Researches in Bio-oriented Industry (BRAIN). Reference: M. Shimono et al. (2003) J Gen Plant Phathol 69:76–82 Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, Japan *National Institute of Agrobilogical Sciences, Tsukuba, Ibaraki 305-8602, Japan ○Masamichi Nishiguchi, Md. Mahfuz, Alam, Akio Miyao*, Hirohiko Hiochika*, Hidemitsu Nakamura*, Hiroaki Ichikawa*, Kappei Kobayashiand Naoto Yamaoka #P870 Functional Characterization of Rice Aspartic Protease (OsAP77) Gene in Response to Bacterial and Fungal Pathogens Fig. Expression patterns of OsAP77 in response to pathogen infections and treatments with signal molecules. The treatments of (A) probenazole (PBZ); (B) M. oryzae with virMo; (C) M. oryzae with avrMo; (D) X. oryzae pv. oryzae with virXoo; (E) wound; (F) SA; (G) INA; (H) H2O2; (I) ABA and (J) MeJA. Numbers represent days after treatment and hours in case of M. oryzae with avrMoand wound. (A) RT-PCR analysis showed that the expression of OsAP77 was induced rapidly by (PBZ), M. oryzae with virMo, M. oryzae with avrMo, X. oryzae pv. oryzae withvirXoo, wound, SA, INA, H2O2, ABA but not by MeJA. OBJECTIVE To know the roles of OsAP77 in response to fungal, bacterial and viral diseases in rice 4. Analysis of progeny of OsAP77 mutant line Fig. Analysis of progeny of OsAP77 mutant line. (A), (B): Genotyping using two sets of primers AP77P-5′/AP77P-3′ and Tos17-5′/AP77P-3′, respectively. Nb, Nipponbare; #1-9, progeny of the mutant line; H2O, negative control. +/+, wild type; +/-, heterozygous; -/- homozygous. (C) The expression level of the OsAP77 in mutant and wild type rice plants by RT-PCR with primers, OsAP-5′/ OsAP-3′. (D) The actin gene was used as the standard control. Tos17 OsAP77 1185 bp Promoter 1999 bp AP77P-5′ Tos17-5′ OsAP-5' OsAP-3′ AP77P-3′ The OsAP77 expression (open reading frame, ORF region, 1185bp) level was highest in WT (+/+) while it was under the detection level in the mutant homozygous (-/-) plants . 5. Symptoms in the knockout mutant lines of OsAP77 Fig. Symptoms in the knockout mutant line of OsAP77 by M. oryzae and X. oryzae pv. oryzae. (A) and (B) Symptoms developed at 7 days after inoculation (dpi) by M. oryzae and X. oryzae pv. oryzae, respectively. (C) and (D), Length of necrotic lesions formed at 7 dpi by M. oryzae and X. oryzae pv. oryzae, respectively. Nb, Nipponbare; Aso, Asominori; Sen, Sensyou; #4 and 6; OsAP77 knock down homogygous (-/-) plants. RESULTS 1. Response to Fungal and bacterial infections -/- +/+ -/- +/+ 4 6 Nb Aso Sen 4 6 Nb Aso Sen Lesion size (mm) A virMo mock Contl avirMo mock Contl B ** Fig. Expression patterns of OsAP77::GUS after inoculation with fungal and bacterial pathogens. A, M. oryzae (virulent race 001, virMo); B, M. oryzae (avirulent race 102.0, avrMo); C, X. oryzae pv. oryzae. D, Rice blast and bacterial leaf blight disease symptoms. ** * * M. oryzae M. oryzae X. oryzae pv. oryzae After the inoculation with either M. oryzae or X oryzae pv. oryzae, the homozygous plants showed more severe symptoms than WT did. Nb Aso Sen 4 Nb Aso Sen 4 6 6 D ** ** CONCLUSION * * The GUS activity was enhanced after inoculation with M. oryzae or X.oryzae pv. oryzae. mock X. oryzae pv. oryzae mock • The transgenic rice plant with GUS reporter gene driven by the promoter of OsAP77, were produced and analyzed. • After M. oryzae or X. oryzae pv. oryzae inoculation, the transgenic plants showed the enhanced GUS activities in vascular tissues surrounding the symptom sites by each pathogen. • An increased level of GUS activity was observed exclusively in vascular tissues after treatment with SA, INA, H2O2 or ABA. • OsAP77 was induced not only by infection with these pathogens,but also after treatment with SA, INA, H2O2 or ABA. • The knockout mutant line of OsAP77 by the insertion of retrotransposon Tos17 showed an enhanced susceptibility compared to wild type in response to M. oryzae, X. oryzae pv. oryzae. These result suggests that OsAP77 have a positive role in defense response to pathogens. virMo mock Contl virMo avirMo mock Vir Xoo 2. Effects of chemical signaling molecules Fig.   Effects of signaling molecules on OsAP77::GUS expression. A, 10 mM SA; B, 5 mM INA; C, 20mM H2O2; D, ABA. The GUS activity was analyzed in the leaves treated with indicated duration (days) after each treatment. The 4th young leaves were cut into 1 cm pieces and used for GUS staining. 0 1 2 3 0 1 2 3 The GUS activity was be induced by PBZ, SA, INA, H2O2 or ABA, which play important roles in signal defense responses.